The ability to site-specifically methylate DNA in vivo (as well as in vitro) would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of cancers and other diseases that involve abnormal hypomethylation of DNA. A site specific DNA methyltransferase would be useful (1) as a tool for studying DNA methylation and the spread of methylation patterns, (2) as a molecular biological tool for silencing genes of interest, and (3) as a potential gene-therapy agent for the selective silencing of genes (e.g. in the treatment of cancers). The molecular tools to carry out truly site-specific control of DNA methylation do not exist. Existing approaches involving the fusion of intact methyltransferases to DNA binding domains at best create methyltransferases with modest preferences for methylation at target sites because the methyltransferases remain active in the absence of the DNA binding domain binding to its target site. The goal of this proposed research is to engineer site-specific DNA methyltransferases that will have the exquisite specificity of methylating unique sites (or clusters of sites) that occur at a frequency of only once per genome. This high degree of specificity will arise from the fact that the methyltransferase will only assemble into an active structure in the context of binding to a target sequence that is long enough to be unique in a genome. A novel combinatorial protein engineering approach coupled with directed evolution will be used to created these site-specific methyltransferases. The research described in this proposal will validate our approach and will be useful in obtaining funding for the long-term goals of developing site-specific methyltransferases that can site alter the methylation pattern at unique sites in a genome in vivo and can alter the expression of a single gene-product in vivo.